Over the past two decades, recombinant DNA technology has led to the commercialization of many protein therapeutics. The most conventional route of delivery for protein drugs has been intravenous (IV) administration because of poor bioavailability by most other routes, greater control during clinical administration, and faster pharmaceutical development. For products that require frequent and chronic administration, the alternate subcutaneous (SC) route of delivery is more appealing. When coupled with pre-filled syringe and autoinjector device technology, SC delivery allows for home administration and improved compliance of administration.
Treatments with high doses of more than 1 mg/kg or 100 mg per dose often require development of formulations at concentrations exceeding 100 mg/ml because of the small volume (<1.5 ml) that can be given by the SC routes. For proteins that have a propensity to aggregate at the higher concentrations, achieving such high concentration formulations is a developmental challenge. Even for the IV delivery route, where large volumes can be administered, protein concentrations of tens of milligrams per milliliter may be needed for high dosing regimens and this may pose stability challenges for some proteins.
The principles governing protein solubility are more complicated than those for small synthetic molecules, and thus overcoming the protein solubility issue takes different strategies. Operationally, solubility for proteins could be described by the maximum amount of protein in the presence of co-solutes whereby the solution remains visibly clear (i.e., does not show protein precipitates, crystals, or gels). The dependence of protein solubility on ionic strength, salt form, pH, temperature, and certain excipients has been mechanistically explained by changes in bulk water surface tension and protein binding to water and ions versus self-association by Arakawa et al in Theory of protein solubility, Methods of Enzymology, 114:49-77, 1985; Schein in Solubility as a function of protein structure and solvent components, BioTechnology 8(4):308-317, 1990; Jenkins in Three solutions of the protein solubility problem, Protein Science 7(2):376-382, 1998; and others. Binding of proteins to specific excipients or salts influences solubility through changes in protein conformation or masking of certain amino acids involved in self-interaction. Proteins are also preferentially hydrated (and stabilized as more compact conformations) by certain salts, amino acids, and sugars, leading to their altered solubility.
Aggregation which requires bi-molecular collision is expected to be the primary degradation pathway in protein solutions. The relationship of concentration to aggregate formation depends on the size of aggregates as well as the mechanism of association. Protein aggregation may result in covalent (e.g., disulfide-linked) or non-covalent (reversible or irreversible) association. Irreversible aggregation by non-covalent association generally occurs via hydrophobic regions exposed by thermal, mechanical, or chemical processes that alter a protein's native conformation. Protein aggregation may impact protein activity, pharmacokinetics and safety, e.g., due to immunogenicity.
A typical approach to minimize aggregation, is to restrict the mobility of proteins in order to reduce the number of collisions. Lyophilization with appropriate excipients may improve protein stability against aggregation by decreasing protein mobility and by restricting conformational flexibility with the added benefit of minimizing hydrolytic reactions consequent to removal of water. The addition of appropriate excipients, including lyoprotectants, can prevent the formation of aggregates during the lyophilization process as well as during storage of the final product. A key parameter for effective protection is the molar ratio of the lyoprotectant to the protein. Generally molar ratios of 300:1 or greater are required to provide suitable stability, especially for room temperature storage. Such ratios can also, however, lead to an undesirable increase in viscosity.
Lyophilization allows for designing a formulation with appropriate stability and tonicity. Although isotonicity is not necessarily required for SC administration, it may be desirable for minimizing pain upon administration. Isotonicity of a lyophile is difficult to achieve because both the protein and the excipients are concentrated during the reconstitution process. Excipient:protein molar ratios of 500:1 will result in hypertonic preparations if the final protein concentration is targeted for >100 mg/ml. If the desire is to achieve an isotonic formulation, then a choice of lower molar ratio of excipient:protein will result in a potentially less stable formulation.
Determining the highest protein concentration achievable remains an empirical exercise due to the labile nature of protein conformation and the propensity to interact with itself, with surfaces, and with specific solutes.
Examples of subjects who may benefit from SC formulations are those that have conditions that require frequent and chronic administration such as subjects with the immune system disease rheumatoid arthritis and immune disorders associated with graft transplantation. Commercially available protein drug products for the treatment of rheumatoid arthritis include HUMIRA®, ENBREL® and REMICADE®.
HUMIRA® (Abbott) is supplied in single-use, 1 ml pre-filled glass syringes as a sterile, preservative-free solution for subcutaneous administration. The solution of HUMIRA® is clear and colorless, with a pH of about 5.2. Each syringe delivers 0.8 ml (40 mg) of drug product. Each 0.8 ml of HUMIRA® contains 40 mg adalimumab, 4.93 mg sodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mg dibasic sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80 and Water for Injection, USP. Sodium hydroxide added as necessary to adjust pH.
ENBREL® (Amgen) is supplied in a single-use pre-filled 1 ml syringe as a sterile, preservative-free solution for subcutaneous injection. The solution of ENBREL® is clear and colorless and is formulated at pH 6.3±0.2. Each ENBREL® single-use prefilled syringe contains 0.98 ml of a 50 mg/ml solution of etanercept with 10 mg/ml sucrose, 5.8 mg/ml sodium chloride, 5.3 mg/ml L-arginine hydrochloride, 2.6 mg/ml sodium phosphate monobasic monohydrate and 0.9 mg/ml sodium phosphate dibasic anhydrous. Administration of one 50 mg/ml prefilled syringe of ENBREL® provides a dose equivalent to two 25 mg vials of lyophilized ENBREL®, when vials are reconstituted and administered as recommended. ENBREL® multiple-use vial contains sterile, white, preservative-free, lyophilized powder. Reconstitution with 1 ml of the supplied Sterile Bacteriostatic Water for Injection (BWFI), USP (containing 0.9% benzyl alcohol) yields a multiple-use, clear, and colorless solution with a pH of 7.4±0.3 containing 25 mg etanereept, 40 mg mannitol, 10 mg sucrose, and 1.2 mg tromethamine.
REMICADE® (Centocor) is supplied as a sterile, white, lyophilized powder for intravenous infusion. Following reconstitution with 10 ml of Sterile Water for Injection, USP, the resulting pH is approximately 7.2. Each single-use vial contains 100 mg infliximab, 500 mg sucrose, 0.5 mg polysorbate 80, 2.2 mg monobasic sodium phosphate, monohydrate, and 6.1 mg dibasic sodium phosphate, dihydrate. No preservatives are present.
Commercially available protein drug products for the treatment of immune disorders associated with graft transplantation include SIMULECT®, and ZENAPAX®.
The drug product, SIMULECT® (Novartis), is a sterile lyophilisate which is available in 6 ml colorless glass vials and is available in 10 mg and 20 mg strengths. Each 10-mg vial contains 10 mg basiliximab, 3.61 mg monobasic potassium phosphate, 0.50 mg disodium hydrogen phosphate (anhydrous), 0.80 mg sodium chloride, 10 mg sucrose, 40 mg mannitol and 20 mg glycine, to be reconstituted in 2.5 ml of Sterile Water for Injection, USP. Each 20-mg vial contains 20 mg basiliximab, 7.21 mg monobasic potassium phosphate, 0.99 mg disodium hydrogen phosphate (anhydrous), 1.61 mg sodium chloride, 20 mg sucrose, 80 mg mannitol and 40 mg glycine, to be reconstituted in 5 ml of Sterile Water for Injection, USP. No preservatives are added.
ZENAPAX® (Roche Laboratories), 25 mg/5 ml, is supplied as a clear, sterile, colorless concentrate for further dilution and intravenous administration. Each milliliter of ZENAPAX® contains 5 mg of daclizumab and 3.6 mg sodium phosphate monobasic monohydrate, 11 mg sodium phosphate dibasic heptahydrate, 4.6 mg sodium chloride, 0.2 mg polysorbate 80, and may contain hydrochloric acid or sodium hydroxide to adjust the pH to 6.9. No preservatives are added
CTLA4Ig molecules interfere with T cell costimulation by inhibiting the CD28-B7 interaction. Therefore, CTLA4Ig molecules can provide a therapeutic use for immune system diseases, such as rheumatoid arthritis and immune disorders associated with graft transplantation.
There is a need for a stable, effective convenient formulations comprising CTLA4Ig molecules for treatment of immune system disorders.